CXC ELR-Positive Chemokines as Diagnostic and Prognostic Markers for Breast Cancer Patients

Simple Summary Currently, breast cancer diagnostics do not have readily available diagnostic and prognostic tools, especially in the early stages of the disease. Therefore, there is a constant need to search for tumor markers that can improve current diagnostics. One such molecule that may be a potential marker could be among a group of chemokines. In this work, we summarize reports that evaluate the expression and peripheral blood concentration of ELR-positive CXC chemokines as potential markers and prognostic factors for breast cancer. Abstract As the most common type of malignant lesison, breast cancer is a leading challenge for clinicians. Currently, diagnosis is based on self-examination and imaging studies that require confirmation by tissue biopsy. However, there are no easily accessible diagnostic tools that can serve as diagnostic and prognostic markers for breast cancer patients. One of the possible candidates for such markers is a group of chemokines that are closely implicated in each stage of tumorigenesis. Many researchers have noted the potential of this molecule group to become tumor markers and have tried to establish their clinical utility. In this work, we summarize the results obtained by scientists on the usefulness of the ELR-positive CXC group of chemokines in ancillary diagnosis of breast cancer.


Introduction
Breast cancer remains the most prevalent cancer in the world. According to WHO data from the GLOBOCAN database, the global incidence in 2020 reached more than 2.2 million new cases, with mortality estimated at around 685,000 [1]. Breast cancer mortality, estimated by the 5-year survival period of patients varies depending on the disease stage at the time of diagnosis. For stage IV breast cancer, survival rates are below 30%, whereas for stage I cases, they are over 99% [2]. These numbers illustrate the importance of early diagnosis in treating breast cancer.
Current diagnostic methods are primarily based on imaging methods such as ultrasound, X-ray, and magnetic resonance imaging (MRI). Implementing mammography screening in many countries for groups of women with an increased risk of breast cancer, that is, around the age of 50, was a breakthrough in streamlining the diagnostic process. Many studies have shown a significant decrease in the risk of breast cancer mortality after introducing mammography into medical practice [3,4]. However, imaging methods also have limitations. A major one is the sheer size of the lesion found in the breast. In the case of microscopic tumors, it is difficult to determine the potential nature of the lesion. Consequently, imaging results must be correlated with a patient's clinical examination and relied on for the histopathologic evaluation of the lesion obtained by tissue biopsy [5]. In current medicine, biopsy evaluation is the only method of confirming a cancerous lesion, which is unfortunately invasive.
Circulating cancer biomarkers have received significant attention in recent years. Venous blood is an easily accessible material, and the collection method has been a routine procedure widely used in disease diagnoses. The most frequently determined blood compounds in diagnosing breast cancer are carcinoma antigen 15-3 (CA 15-3), carcinoembryonic antigen (CEA), or carcinoma antigen 27.29 (CA 27.29). However, it should be noted that these molecules have limited sensitivity and specificity, which results in their unsuitability for screening [6]. Due to the limited utility of current markers in the ancillary diagnosis of breast cancer, there is a search for new compounds whose assays could be an adjunctive tool in the diagnosis of this condition. Such compounds could be chemokines [7]. Chemokines in the body form and maintain the immune response, engage in the process of angiogenesis, and determine the chemoattraction and migration of cells that possess chemokine receptors. As a result, these compounds may be involved in each phase of the tumorigenesis process, from the tumor's initiation and proliferation by local dissemination up to its spread in distant locations. There have also been reports of chemokines' role in driving resistance to applied treatments [7,8]. For these reasons, chemokines may be helpful in the diagnostic process and act as potential candidates for tumor markers. Apart from circulating molecules' blood concentrations, many researchers also note that their elevated or reduced tissue expression may have prognostic significance for the course of treatment. They may also correlate with lymph node involvement, the occurrence of distant metastasis, or at least be associated with a specific molecular subtype of breast cancer. This yet-to-be-studied area brings great, but still rather dim, hopes for advancing the diagnostic and prognostic evaluation of breast cancer. In this paper, we introduce and describe the potential use of the ELR-positive CXC group of chemokines in ancillary diagnosis of breast cancer.

Breast Cancer: A Brief Overview
Breast cancer is a heterogeneous disease. It has high histopathological and molecular diversity, as well as various degrees of cell differentiation. Breast cancer often manifests itself as a palpable lesion in the breast. Other less common symptoms include tightening or pulling of the skin over the lesion, retraction or leakage of the nipple, or, less commonly, a change in breast size, skin color, or enlargement of the axillary lymph nodes (including lymph node engagement with the simultaneous absence of symptoms originating from the breast itself). Such symptoms can simultaneously occur with benign breast lesions; therefore, imaging and histological examinations based on fine-or core-needle biopsy are necessary for identification [9].
Based on the histological type, breast lesions are divided into benign tumors, lesions of an indeterminate, borderline, or uncertain nature, carcinoma in situ, or G3 intraepithelial neoplasia, and malignant tumors with distant metastatic foci [10,11] (Supplementary  Table S1). Malignant breast cancers are classified into two major groups: carcinomas and sarcomas. Carcinomas are the most common breast tumors and originate from the epithelial cells of the breasts' lobules or ducts [11]. Breast cancer can be classified molecularly or by staging systems. Particularly relevant is the molecular classification that divides breast cancer into four major subtypes: Luminal A, Luminal B, HER2-enriched (HER2+), and triple-negative breast cancer (TNBC) [12,13]. Segregation into each subtype is based on the expressions of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and cell proliferation factor (Ki-67) [14].
The pathogenesis of breast cancer is a complex process that remains incompletely studied. Although much is known about risk factors and protective factors, some aspects are still controversial. A diagram containing various factors is shown in Figure 1.
The biochemical diagnosis of breast cancer has mostly supportive applications and generally uses cancer antigen 15-3 (CA 15-3), cancer-embryonic antigen (CEA), and cancer antigen 27.29 (CA 27.29) [15][16][17][18]. However, the previously mentioned low sensitivity and specificity of these compounds preclude their use in screening [19,20]. In addition, these markers have low concentrations at the early stages of breast cancer, which decreases their screening utility. Importantly, serum concentrations of CA 15-3, CEA, and CA 27.29 are affected by several pathological phenomena that coexist with cancerous lesions in the breast. These include, among others, liver, lung, and endocrine diseases [21][22][23][24]. However, the aforementioned compounds have been found to have clinical utility in monitoring the effectiveness of therapy and detecting early recurrence or the presence of metastases, especially in cases where radiological diagnoses cannot be used [6].

An Outline of the CXC Chemokine Group
CXC chemokines belong to a family of chemotactic cytokines comprising around 50 proteins. They consist of four cysteine residues forming two disulfide bridges. The first two cysteine residues are separated by a single amino acid. As previously mentioned, chemokines are involved in controlling many processes, both physiological and pathological. One of their most important functions is regulating immune balance and directing the immune response [28,29]. In addition, CXC group chemokines are among the key elements regulating inflammatory and angiogenic processes. Depending on whether a 3-amino-acid glutamine-leucine-arginine motif (ELR motif) is present at the N-terminus of the chain, the CXC group of chemokines is divided into two subgroups: ELR+ promoting angiogenesis and ELR-with angiostatic properties. It is important to note that CXCL12, which belongs to the ELR-subgroup, is an exception to ELR motif dependence for angiostatic properties, and is instead involved in promoting angiogenesis through its interaction with receptors.
Three mechanisms may involve chemokines in tumorigenesis: 1.
Control of angiogenesis-Allows tumor growth and metastasis by providing easy access to oxygen and nutrients.

2.
Immune regulation-Controls the influx of leukocytes into the tumor microenvironment.

3.
Modification of the functioning of cancer cells-Interacts with chemokine receptors and triggers intracellular signaling pathways.
These mechanisms can act in a procarcinogenic manner, e.g., by stimulating angiogenesis, the influx of lesion-promoting leukocytes, or altering the biological profile of the cell (increased proliferation, avoidance of apoptosis, and decreased adhesion to the microenvironment). However, they can also act in an anticarcinogenic manner by inhibiting angiogenesis and stimulating non-specific anti-tumor immunity through an influx of leuko-cytes [8,[28][29][30][31]. Dysregulation of chemokine expression in the tumor microenvironment can also direct tumor cells to other locations and promote their spread in the body.
The tumor microenvironment and its components, including growth factors [32][33][34], chemokines [33,[35][36][37], or matrix metalloproteinases [38][39][40], are of scientific interest for their potential diagnostic and prognostic factors and as potential therapeutic targets. The breast tumor stroma, however, is abundant in fibroblasts, which are one of the main sources of chemokines. Other sources of these molecules include leukocytes, epithelial, endothelial, and mesenchymal cells, including tumor-altered cells [7,28,41,42]. Therefore, focusing on this group of molecules in the breast cancer diagnostic process seems natural.

CXC ELR-Positive Chemokines as Biomarkers of Breast Cancer
The issues discussed in the current chapter are summarized in Table 1 at the end of the article.
Part of the findings were also related to data obtained from blood tests. Ma et al. [36] noted higher CXCL1 serum levels among breast cancer patients than healthy women (p = 0.011). In our own study, we did not compare the CXCL1 plasma concentrations between luminal breast cancer patients and healthy women [35]. Moreover, in women with benign breast lesions, plasma CXCL1 concentrations also remained at similar levels to cancer patients and healthy women [35]. High plasma levels of CXCL1 along with TGF-β in patients with metastatic breast cancer were also associated with increased detection of circulating tumor cells (TGF-β p < 0.0001; CXCL1 p = 0.02) and shorter overall survival (CXCL1 p = 0.05; CXCL1+ TGF-β p = 0.001) [33].
Li et al. [60] obtained similar results when serum concentrations were determined. Higher concentrations were obtained in breast cancer patients than in the serum of healthy controls (p < 0.001) [60]. Li et al. also determined the potential of serum CXCL5 concentrations as a diagnostic marker by obtaining a diagnostic sensitivity of 65.3%, a diagnostic specificity of 60%, and a diagnostic test power of AUC = 0.6970 [60]. Wang et al. also studied the serum concentration of CXCL5 and found that it did not differ between breast cancer patients and patients with benign and proliferative breast lesions. However, the concentration differed by tumor size (p < 0.001) and positively correlated with Ki-67 expression levels (p = 0.027) [37].

CXCL6 and CXCL7
Bièche et al.'s study of CXCL6 mRNA expression in breast cancer, as well as data analyses from the TCGA, Oncomine, and Genotype-Tissue Expression databases, showed no differences between tumor and physiological breast tissues [48,49,57]. However, expression was higher in the TNBC molecular subtype than in ER+ subtypes (p < 0.05) [48]. However, Chen et al. and Hozhabri et al. showed significantly lower CXCL6 expression in breast cancer than in normal tissues (p < 0.05) [47,50]. They noted that the CXCL6 expression level was significantly higher in metastatic cancer cells and grade 3 tumor cells than in low graded cells but showed no correlation with overall survival (p < 0.05 [57]; p < 0.001 [50]). Chen et al. and Hozhabri et al. also noticed this lack of correlation but managed to show a positive correlation between CXCL6 expression and relapse-free survival (p < 0.001 [47]; p = 0.00012 [50]). On the other hand, other studies revealed that CXCL6 overexpression was associated with longer overall survival (p = 0.036) [48,49].
Wang et al. [37] found that CXCL7 serum levels were negatively correlated with Ki 67 expression levels (p = 0.042). However, serum levels did not differ between malignant, benign, and proliferative breast lesions [37]. Kosir and Ju [62] also examined CXCL7 serum levels and noted that they were significantly higher in breast cancer patients than in healthy individuals (p < 0.05). Based on 23 pairs of serous specimens, they also assessed that, relative to preoperative levels, postoperative CXCL7 levels decreased significantly (p < 0.05) and reached levels comparable to healthy controls [62].
Several scientific papers have also reported increased serum CXCL8 (p = 0.047 [66]; p < 0.001 [67]; p < 0.001 [68]) or plasma levels (p < 0.001 [52]; p = 0.005 [35]) in patients with early (p = 0.002) and advanced stages (p = 0.001) of breast cancer [69] compared with healthy women. Additionally, higher concentrations relative to healthy controls were shown for the ER+ (p = 0.021), PR+ (p = 0.039), and TNBC (p = 0.046) subgroups of breast cancer [68]. Women with benign breast lesions also showed higher levels of CXCL8 than healthy women (p < 0.001 [52]; p = 0.033 [35]; p < 0.001 [68]), but only one paper showed significant differences between malignant and benign lesions (p < 0.001 [68]). Wang et al. also noted significant differences between benign lesions and in situ versus invasive cancer (p = 0.006), but no post hoc evaluation was performed to define the differences between the groups [37]. However, they did evaluate the differences between the in situ group and the invasive type, obtaining higher concentrations for the in situ type (p = 0.002). Based on the results of binary logistic regression analysis, they found that serum CXCL8 concentration was a predictor of differentiation between the benign lesion group (p = 0.024), breast cancer group (p = 0.011), and healthy controls [37]. According to these results, CXCL8 may be a useful diagnostic marker for breast cancer, which we also investigated in a different paper. CXCL8 concentration showed higher sensitivity (70%), positive predictive value (77.78%), negative predictive value (50%), diagnostic test power (AUC = 0.6410), and similar specificity (60%) than CA 15-3 (55%; 75.34%; 41.56%; AUC = 0.6300; 64%, respectively) [35]. A panel of CXCL8 and CA 15-3 combined increased the sensitivity of the test to 88%, the negative predictive value to 61.29%, and the diagnostic power of the test to AUC = 0.6582 with a high positive predictive value (73.95%) and a decrease in specificity (38%) [35]. Khalaf et al. also evaluated the diagnostic potential of CXCL8 levels. CXCL8 concentration differentiated a group of breast cancer patients from healthy controls with 95.6% sensitivity, 95% specificity, and a diagnostic test power equal to AUC = 0.998. In a test to differentiate benign lesions from healthy individuals, CXCL8 achieved sensitivity, specificity, and diagnostic test power equal to 82.1%, 75%, and AUC = 0.804 [68], respectively. A combination of three chemokines, CXCL8, CXCL9, and CCL22, was evaluated by Narita et al. as a discriminating tool for healthy individuals and those with breast cancer, obtaining a high value of AUC = 0.7771 [52]. These papers vary notably in their suggested diagnostic utility. However, the size of the groups and their specification should be considered. In our work [35], we focused exclusively on luminal subtypes of breast cancer, where the final count of breast cancer patients was 100 and 50 in the group of women with benign lesions and controls, respectively. By contrast, Khalaf et al. studied [68] 45 women with breast cancer, 25 women with benign lesions, and 20 healthy controls. The 45 women with breast cancer included all possible molecular subtypes of breast cancer, which, given the differences in concentration levels based on receptor expression, may have been a key impact on the results.

Conclusions
Breast cancer is the most common malignancy affecting women. This disease is molecularly diverse but has a good prognosis if detected early enough. It is estimated that the 5-year survival rate of patients with stage I breast cancer is as high as 99%. A breast cancer diagnosis is primarily based on imaging examinations such as ultrasound, X-ray, or MRI, which have numerous limitations. Furthermore, the obtained results are confirmed invasively via biopsy. These methods translate into a longer diagnosis time, thus worsening the prognosis for women. There is considerable hope in new ancillary breast cancer diagnosis methods, which include the non-invasive determination of tumor markers from peripheral blood. Potential markers for breast cancer diagnosis include chemokines. This article summarizes existing knowledge about the roles of CXCL ELR-positive chemokines CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8 as peripheral blood and tissue markers in the diagnosis and prognosis of women with breast cancer.
Chemokines have been shown to mediate the pathogenesis of breast cancer. However, data on their potential use in diagnosing the disease are conflicting or contradictory. Although all CXCL ELR-positive chemokines are expressed in cancerous breast tissue, their expression may be elevated or downregulated compared to normal tissue. In addition, the mRNA levels of these chemokines are not always associated with the clinical features of breast cancer, or the data obtained by different research teams are mutually exclusive. We reached similar conclusions after analyzing data on CXCL ELR-positive chemokines' potential as markers in peripheral blood. Nevertheless, several compounds in this group show considerable potential as tissue or peripheral blood markers.
Establishing the unequivocal importance of CXCL ELR-positive chemokines in breast cancer requires more research. However, we believe that several compounds in this group show high potential. Based on our analysis of accumulated data, CXCL8 has preliminary high potential as a new diagnostic and prognostic marker, while CXCL2 and CXCL6 can be considered as prognostic markers. In the case of CXCL1 and CXCL5, however, we cannot determine their usefulness due to insufficient information.
Regarding TNBC, characterized by a particularly unfavorable prognosis, CXCL1, CXCL2, CXCL3, and CXCL8 are found to be strongly overexpressed relative to luminal types of breast cancer, suggesting that these chemokines can be used to differentiate cancer subtypes or as potential indirect therapeutic targets. The first studies of targeted therapies against individual chemokines did not show the expected efficacy [73,74] due to the multiligand character of their receptors. However, targeting the receiving point in the ligand-receptor signaling axis or using modified immune cells in therapy may result in the expected therapeutic effect, which may, unfortunately, result in severe side effects on the organism's healthy cells and tissue [75,76]. Nevertheless, new strategies such as modified cell therapy are worth the interest and may signify a breakthrough in treating breast cancer, especially the TNBC subtype.  Table S1: Histological classification of breast tumors. References [9,10]